Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-12-21
2001-08-14
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S019300, C514S021800, C514S784000, C514S822000, C514S970000, C530S324000, C530S855000
Reexamination Certificate
active
06274553
ABSTRACT:
TECHNOLOGICAL FIELD OF THE INVENTION
The present invention relates to a method for stabilizing and controlling change in a peptide which comprises a sequence -Asp-Gly- or -Asn-Gly- in the amino acid sequence, wherein the sequence -Asp-Gly- or -Asn-Gly- changes into a succinimide moiety by a dehydration reaction or a deamidation reaction and further changes into a &bgr;-rearranged moiety by an isomerization reaction, particularly to a method for controlling such a change in a peptide such as desulfatohirudin or a hirudin variant. The present invention also relates to a lyophilized pharmaceutical composition containing a peptide for which the change is controlled by this stabilizing method.
BACKGROUND OF THE INVENTION
Hirudin is an anti-blood coagulation factor which is secreted from the salivary glands of Hirudo medicinal is. Since hirudin exhibits an anti-thrombin activity, this compound and its variants are used as anti-blood coagulation drugs.
The hirudin and most hirudin variants contain a sequence of -Asp-Gly- or -Asn-Gly- and change into succinimide compounds due to a change in this sequence with the passage of time. The succinimide compounds further change into &bgr;-rearranged compounds. Because of this, even if the compounds are sufficiently purified in the production line, the purity of the compounds decreases with the passage of time due to production of the succinimide compounds and &bgr;-rearranged compounds. Because the succinimide compounds and &bgr;-rearranged compounds exhibit anti-thrombin activity themselves (Japanese Patent Application Laid-open No. 310788/1993), such a decrease in purity does not cause serious problems in general. However, such deterioration of the purity is not desirable when these compounds are used as drugs. Therefore, various studies have been undertaken to improve the stability of hirudin.
Examples of them include a method of adding a water-soluble salt of calcium and/or magnesium to hirudin to increase the stability of hirudin (Japanese Patent Application Laid-open No. 267877/1995), a method of adding potassium phosphate and sugar (WO 95/20399), and the like. However, these proposed methods cannot accomplish sufficiently increase in the stability of hirudin.
DISCLOSURE OF THE INVENTION
The present invention has been achieved to overcome these problems.
Specifically, an object of the present invention is to provide a method for stabilizing a peptide containing a sequence -Asp-Gly- or -Asn-Gly-, which comprises controlling change of the sequence -Asp-Gly- or -Asn-Gly- into a succinimide moiety or a &bgr;-rearranged moiety.
Another object of the present invention is to provide a hirudin-containing lyophilized pharmaceutical composition with excellent stability by utilizing this stabilizing method.
The above object is achieved in the present invention by a stabilization method comprising:
providing a solution of a peptide which contains a sequence -Asp-Gly- or -Asn-Gly- which may be converted into a succinimide moiety shown by the following general formula (1),
or a &bgr;-rearranged moiety shown by general formula (2),
adding an organic acid and adjusting the pH of the solution to 5-6.5, and
lyophilizing the resulting mixture.
Desulfatohirudin or a hirudin variant is used as the peptide in the present invention.
The above object is further achieved in the present invention by a pharmaceutical composition prepared using the above-mentioned stabilization method of peptide, specifically by a hirudin-containing lyophilized pharmaceutical composition comprising 5 to 100 mol of an organic acid added to one mol of desulfatohirudin or a hirudin variant. In a preferred embodiment of the present invention, in addition to the organic acid, 1 to 500 mol of saccharose and, as required, 10 to 1000 mol of mannitol, for one mol of desulfatohirudin or a hirudin variant, are added to the peptide solution to be lyophilized.
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Furuya Hideyuki
Michibuchi Kose
Morita Hiroyuki
Takatsu Yukitaka
Tanigawa Makoto
Japan Energy Corporation
Low Christopher S. F.
Mohamed Abdel A.
Testa Hurwitz & Thibeault
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